1. Field of the Invention
The present invention relates generally to the field of cytokines. More particularly, it concerns CXC chemokines, CXC chemokine analogues, and methods of using such chemokines, for example, in modulating angiogenic and angiostatic responses.
2. Description of the Related Art
Cytokines are, generally, small protein or polypeptide-based molecules that modulate the activity of certain cell types following binding to cell surface receptors. The CXC (xcex1) chemokines are one group of cytokines, so named due to the conserved Cys Xaa Cys sequence element located towards their N-terminus. The CXC chemokine family includes interleukin-8 (IL-8); xcex3-interferon-inducible protein-10 (IP-10); Platelet Factor 4 (PF4); the growth related oncogene (GRO) peptides GROxcex1, GROxcex2 and GROxcex3; monokine induced by gamma-interferon (MIG); epithelial neutrophil activating protein-78 (ENA-78); granulocyte chemotactic protein-2 (GCP-2); and the NH2-terminal truncated forms of platelet basic protein (PBP), namely connective tissue activating protein-III (CTAP-III), xcex2-thromboglobulin (xcex2TG) and neutrophil activating peptide-2 (NAP-2).
IL-8 is a peptide of approximately 8 kD, and is about 72 amino acids in length, with this length varying according to the post-translational processing in different cell types (Yoshimura et al., 1989; Strieter et al., 1989b). The IL-8 gene was initially identified by analyzing the genes transcribed by human blood mononuclear cells stimulated with Staphylococcal enterotoxin A (Schmid and Weissman, 1987). IL-8 production is induced by tumor necrosis factor and by interleukin-1 (Strieter et al., 1989a; 1989b; 1990a).
The first biological roles of IL-8 to be defined were those connected with its ability to stimulate neutrophil chemotaxis and activation (Yoshimura et al., 1987a; Schroder et al., 1988; Peveri et al., 1988; Larsen et al., 1989). If neutrophils are xe2x80x98primedxe2x80x99, e.g., by E. coli endotoxin (also known as lipopolysaccharide or LPS), IL-8 also stimulates the neutrophil to release certain enzymes, such as elastase and myeloperoxidase.
Physiologically, high concentrations of IL-8 have been connected with inappropriate neutrophil activation and certain disease conditions, such as adult respiratory distress syndrome (ARDS) (Miller et al., 1992; Donnelly et al., 1993); rheumatoid arthritis (Brennan et al., 1990; Koch et. al., 1991a; Seitz et al., 1991); pseudogout (Miller and Brelsford, 1993); and cystic fibrosis (McElvaney et al., 1992; Nakamura et al., 1992; Bedard et al., 1993). It has also been reported that IL-8 participates in inflammatory processes in the eye that may contribute to tissue destruction (de Boer et al., 1993; Ferrick et al., 1991; Wakefield and Lloyd, 1992) and that IL-8 is involved in corneal neovascularization (Strieter et al., 1992a).
IP-10 is an interferon-inducible chemokine, the exact function(s) of which have yet to be elucidated (Luster et al., 1985). It is believed that IP-10 may have a role in cellular immune and inflammatory responses (Luster and Ravetch, 1987a). IP-10 has been reported to exert an anti-tumor effect in vivo, but not in vitro (Luster and Leder, 1993). The mechanism underlying the in vivo anti-tumor effects was suggested to involve T cell recruitment, and, more specifically, to likely be a result of secondary T cell products (Luster and Leder, 1993).
Information concerning the nucleic acids encoding IL-8 has been available for a number of years (e.g., Lindley et al., 1988; Schmid and Weissmann, 1987; Matsushima et al., 1988; Hxc3xa9bert et al., 1991). Truncated and genetically engineered variants of IL-8 have also been described (Moser et. al., 1993; Baggiolini et al., 1994). IP-10-encoding sequences are also available (Luster et al., 1985; Luster and Ravetch, 1987b). Furthermore, the genomic organization of IL-8 and IP-10 has now been analyzed (Mukaida et al., 1989; Modi et al., 1990; Luster et al., 1987; Luster and Ravetch, 1987a; Kawahara and Deuel, 1989).
PF4 was originally identified for its ability to bind to heparin, leading to inactivation of heparin""s anticoagulation function (Deutsch and Kain, 1961). PF4 was later reported to be capable of attenuating the growth of murine melanoma and human colon cancer (Sharpe et al., 1990). The three dimensional structure of PF4 has been reported (St. Charles et al., 1989). MIG is a CXC chemokine that appears to be only expressed in the presence of xcex3-interferon (xcex3-IFN) (Farber, 1993).
ENA-78 and GCP-2 were initially identified on the basis of their ability to induce neutrophil activation and chemotaxis (Walz et al., 1991; Baggiolini et al., 1994). GCP-2 has been more recently studied by Proost et. al. (1993a; 1993b). NAP-2, CTAP-III (and xcex2TG) are proteolytic cleavage products of PBP (Walz and Baggiolini, 1990). The xcex2TG structure has been described by Begg et al. (1978).
GROxcex1, GROxcex2, and GROxcex3, are closely related CXC chemokines, with GROxcex1 originally described for its melanoma growth stimulatory activity (Anisowicz et al., 1988). GROxcex1 is also termed MGSA; GROxcex2 is also termed MIP-2xcex1; and GROxcex3 is also termed MIP-2xcex2 (Wolpe et al., 1988). GRO peptides have been proposed to contribute to would healing in vitreoretinopathy (Jaffe et al., 1993). GRO genes have been reported to be over-expressed at sites of injury and neovascularization, and are said to be important in would healing (Martins-Green et al., 1990, 1991; Iida and Grotendorst, 1990). However, a review of the scientific literature shows that the functions of the GRO genes have yet to be clearly defined, with roles in negative growth regulation, alteration of the extracellular matrix and in cell cycle control being proposed (Anisowicz et al., 1988; Martins-Green et al., 1990, 1991).
As mentioned above, one of the well documented actions of IL-8 at the cellular level is that it activates neutrophils, as assessed by the induction of neutrophil chemotaxis and enzyme release. However, certain other CXC chemokines, including PF4, are reported to be virtually inactive towards neutrophils (Walz et al., 1989). IL-8 is believed to bind to two different receptors on neutrophils, whereas other chemokines seem to bind to only one receptor (Holmes et al., 1991; Murphy and Tiffany, 1991; LaRosa et al., 1992, Cerretti et al., 1993). The IL-8 receptors are coupled to GTP-binding proteins (G proteins), allowing transmission of the IL-8 signal into the cell (Wu et al., 1993).
The three dimensional structure of IL-8 has been elucidated by NMR (Clore et al., 1990) and by X-ray crystallography (Clore and Gronenborn, 1992; Baldwin et al., 1991). A freely movable amino terminal end is followed by three beta pleated sheets and an alpha helix is located at the carboxyl-terminal end (Oppenheim et al., 1991). Despite the structural information available, there are several lines of conflicting evidence regarding which portions of the IL-8 polypeptide mediate receptor binding. From the literature, it seems that both the amino- (Clark-Lewis et al., 1991a; Moser et al., 1993) and carboxyl-terminal ends (Clore et al., 1990) may be involved in IL-8 binding to its receptors.
The issue of the precise function of IL-8 receptors on neutrophils appears to be further complicated by the fact that certain neutrophil receptors also bind to other CXC chemokines, particularly NAP-2 and GROxcex1 (Moser et al., 1991). However, in studying NAP-2 and IL-8, Petersen et. al. (1994) reported that although these cytokines bind to the same sites on neutrophils, they interact in different ways. Particular discrepancies in binding affinities, receptor densities and biological effects were reported, leading the authors to conclude that these CXC chemokines could mediate different biological functions by interacting with common receptors, but in an individual manner (Petersen et. al., 1994).
The amino acid sequence ELR (Glu Leu Arg) located within IL-8, and found within the N-terminus of certain other CXC chemokines, has been proposed to be involved in IL-8 receptor binding to neutrophils. The ELR motif of IL-8 has thus been proposed to be involved in mediating certain of the biological functions of IL-8, particularly neutrophil activation (Hxc3xa9bert et al., 1991; Clark-Lewis et al., 1991b; 1993; Moser et al., 1993). In this regard, Clark-Lewis et al. (1993) reported that adding the ELR motif to PF4 allowed the resultant modified PF4 to bind to IL-8 receptors and to activate neutrophils.
Following the Clark-Lewis et al. (1993) studies, it appears to be generally accepted that the N-terminal ELR motif is important for IL-8 binding to certain well-characterized receptors, and that ELR is required for certain of the IL-8 biological activities, namely neutrophil attraction, activation, chemotaxis and enzyme release. The ELR motif is absent in molecules such as PF4 and IP-10, which may explain why these molecules are devoid of neutrophil binding and attracting activities (Baggiolini et al., 1994).
However, as a caveat to the above ELR-PF4 data, even the same studies by the Clark-Lewis group resulted in the finding that adding the ELR motif to IP-10 and to the CC chemokine, monocyte chemoattractant protein-1 (MCP-1), did not impart neutrophil-activating properties to these chemokines. This led Clark-Lewis to the conclusion that the ELR motif was necessary, but not sufficient, for IL-8 receptor binding and neutrophil activation (Clark-Lewis et. al., 1993). These authors also implied that other regions of the IL-8 protein are important for neutrophil activation (Clark-Lewis et al., 1993); and later stated that additional structural requirements need to be identified for the design of inhibitors with potential therapeutic applications (Moser et. al., 1993).
Furthermore, other differences do exist between IL-8 and PF4 that may account for differential receptor binding and biological properties. For example, Clore et. al. (1990) proposed that the distribution of positively-charged residues in the 59-67 amino acid region may be an important determining factor in recognition and activity.
Naturally occurring chemokines that are inactive towards neutrophils have not been reported to possess antagonistic activity against chemokines that exhibit chemotactic and activating activity towards these cells. However, Moser et al. (1993) described certain synthetic IL-8 analogues that inhibited IL-8-mediated neutrophil responses and that qualify, in certain terms, as IL-8 antagonists. However, in these studies it was found that even a single IL-8 derivative would exert differential effects on various neutrophil responses, such as chemotaxis, exocytosis and respiratory burst (Moser et al., 1993).
Additional published papers have suggested that further diverse structural elements are required for CXC chemokine actions. For example, one group of workers reported that changing Tyr28 and Arg30 in MCP-1 results in an IL-8-like molecule, and hypothesized that one or both of these residues are important for cytokine-receptor binding (Beall et al., 1992). Brandt et. al. (1993) identified a novel molecular variant of NAP-2 that had enhanced biological activity. This variant was found to be truncated at the C-terminus, lacking from one to three amino acid residues, and these authors suggested that proteolytic modification at the C-terminus plays a role in the regulation of NAP-2-biological activity (Brandt et. al., 1993).
It has also been shown that structurally similar CXC chemokines, such as NAP-2 and CTAP-III (each derived from the same precursor), have markedly different activities towards neutrophils (Walz et al., 1989). Proost et. al. (1993a) also showed that although GCP-2 is structurally related to IL-8 and GROxcex1, it has different biological actions. Therefore, there does not appear to be a consensus in the art as to the important functional regions present even within the IL-8 primary structure, let alone an agreement as to important functional regions in all CXC chemokines.
It has further been reported that certain CXC chemokines have angiogenic functions. An example of an angiogenic CXC chemokine is IL-8, which induces angiogenesis in ex vivo models and in vivo (Koch et al., 1992b; Strieter et al., 1992a; Hu et al., 1993). Antibodies against IL-8 and IL-8 antisense oligonucleotides block the angiogenic activity of IL-8 (Koch et al., 1992b; Smith et al., 1994). Anti-IL-8 strategies have thus been proposed as a potential means for treating cancer (Smith et al., 1993; Burdick et al., 1994; Smith et al., 1994). One CXC chemokine, PF4, has been described as having angiostatic properties (Maione et al., 1990) and, in another study, has also been reported to inhibit the growth of certain cancers (Sharpe et al., 1990).
In contrast to the information concerning the action of CXC chemokines on isolated neutrophils, their actions on other cell types and their actions in vivo have not been well defined in many cases. Although useful in that particular field of study, the data regarding neutrophil activation is not particularly relevant to the complex issues of angiogenesis. In fact, there is a significant lack of data concerning the angiogenic or angiostatic properties of the CXC chemokines. For example, there is little, if any, information on the types of receptors, or even on the types of cells, that may be responsible for mediating the ultimate effects of CXC chemokines on the vasculature. As to the regions of these molecules that are believed to exert such effects, the teaching in the art appears to be particularly confused.
For example, two groups of workers proposed the angiostatic site of PF4 to be the heparin-binding site of the molecule, i.e., to lie within the C-terminus (Maione et al., 1990; Sharpe et al., 1990; Han et al., 1992). However, one of these groups (Maione et al., 1991) later distinguished the angiostatic site from the heparin-binding site by using a non heparin-binding PF4 analogue. Maione et al. (1991) then proposed that the angiostatic site was located in another, distinct region of the C-terminal part of the molecule.
There are no reports in the literature indicating that angiostatic chemokines can inhibit the angiogenic activity of angiogenic chemokines or other angiogenic cytokines (e.g., bFGF). In addition, the literature does not contain any examples that describe the introduction of angiogenic activity to angiostatic chemokines or the introduction of angiostatic activity to angiogenic chemokines through the manipulation of the chemokines"" structures. Thus, there is no definitive information in the prior art as to the important functional regions in the angiogenic and angiostatic chemokines. As the CXC chemokines are involved in important physiological processes, it is evident that a more precise understanding of the structural elements that control their biological activity is needed.
The present invention, in a general and overall sense, concerns the inventors"" discovery that CXC chemokines having the ELR (Glu Leu Arg) motif are angiogenic and those lacking the ELR motif are angiostatic. Although now seen to be elegantly simple, this discovery represents a marked advance over the confused and conflicting teachings of the prior art and provides new uses of the CXC chemokines and other molecules.
A particular feature of this invention is the identification of two angiostatic agents, namely IP-10 and MIG, each of which lack the ELR motif. These known CXC chemokines have not been previously identified as having angiostatic activity. The characterization of CXC chemokines according to the absence of the newly-discovered angiogenic motif was followed by the generation of experimental data confirming the angiostatic activity of IP-10, MIG and CXC chemokines modified to remove the ELR motif. The angiogenic activity of previously poorly-characterized CXC chemokines, such as GCP-2, was then established, as was the angiogenic activity of MIG modified to include the ELR motif.
The discovery that IP-10 and MIG block ELR CXC chemokine-induced angiogenesis could not be expected from a study of the published scientific literature, particularly as IP-10 and MIG are known not to block CXC chemokine-induced neutrophil activation (DeWald et. al., 1992). The present invention allows, for the first time, angiogenic or angiostatic chemokines to be identified or designed without laborious experimentation and avoiding the expense of trial and error screening.
As used herein, the term CXC chemokine is used to refer to a cytokine that has the amino acid motif Cys Xaa Cys located in the N-terminal region, or a derivative or mutant thereof. Examples of CXC chemokines include IL-8; ENA-78; GCP-2; GROxcex1 (MGSA), GROxcex2 (MIP-2xcex2) and GROxcex3 (MIP-2xcex2); CTAP-III; NAP-2; xcex2TG; IP-10; MIG and PF4. For simplicity, the terms xe2x80x9cCXC chemokinexe2x80x9d and xe2x80x9cCXC chemokine compositionxe2x80x9d are used to describe both the native or wild type chemokines and those CXC chemokines with sequences altered by the hand of man (engineered chemokines).
A CXC chemokine that contains the amino acid motif ELR is referred to as an xe2x80x9cELR-CXC chemokine (ELR-CXC)xe2x80x9d, whereas a CXC chemokine that does not contain this motifis be termed an xe2x80x9cXXX-CXC chemokinexe2x80x9d, or referred to as XXX-CXC. Examples of ELR-CXC chemokines include IL-8, ENA-78, GCP-2, GROxcex1, GROxcex2, GROxcex3, CTAP-III, NAP-2 and xcex2TG. Examples of XXX-CXC chemokines include IP-10, MIG and PF4.
The term xe2x80x9cwild typexe2x80x9d CXC chemokine refers to those ELR-CXC or XXX-CXC chemokines that have an amino acid sequence as found in the chemokine in the natural environment. This term therefore refers to the sequence characteristics, irrespective of whether the actual molecule is purified from natural sources, synthesized in vitro, or obtained following recombinant expression of a CXC chemokine-encoding DNA molecule in a host cell.
The terms xe2x80x9cmutant, variant or engineeredxe2x80x9d CXC chemokine refer to those ELR-CXC or XXX-CXC chemokines the amino acid sequence of which have been altered with respect to the sequence of the chemokine found in nature. This term thus describes CXC chemokines that have been altered by the hand of man, irrespective of the manner of making the modification, e.g., whether recombinant DNA techniques or protein chemical modifications are employed.
xe2x80x9cNativexe2x80x9d CXC chemokines are those that have been purified from their natural sources, such as from tissues or from cultured, but otherwise unaltered, cells. Native CXC chemokines will also most generally have wild type sequences.
xe2x80x9cRecombinantxe2x80x9d CXC chemokines are those molecules produced following expression of a CXC chemokine recombinant DNA molecule, or gene, in a prokaryotic or eukaryotic host cell, or even following translation of an RNA molecule in an in vitro translation system. xe2x80x9cSyntheticxe2x80x9d CXC chemokines are those chemokines produced using synthetic chemistry, most usually in the form of automated peptide synthesis. Both recombinant and synthetic CXC chemokines may have either wild type or mutant sequences, as designed.
The preparation of wild type, mutant, native, recombinant and synthetic CXC chemokines will be straightforward to those of skill in the art in light of the present disclosure. Techniques for the operation of automated peptide synthesizers, and for the expression of recombinant proteins (see e.g., Sambrook et al., 1989) are standard practice in the art and are further described herein in detail. To prepare a recombinant CXC chemokine composition, all that is required is to express the CXC chemokine gene, including wild type and mutant genes, in a recombinant host cell and to collect the expressed CXC chemokine protein to obtain the composition.
In certain embodiments, the present invention concerns methods for inhibiting angiogenesis. These methods generally comprise administering to an animal a biologically effective amount of a CXC chemokine composition, preferably a pharmaceutically acceptable composition, that comprises, or results in the production of, an XXX-CXC chemokine other than PF4. XXX-CXC chemokines include IP-10, MIG and ELR-CXC chemokines that have been modified to remove the amino acid sequence ELR, and combinations of such agents. xe2x80x9cBiologically effective amountsxe2x80x9d are those amounts that function to inhibit or reduce angiogenesis in the animal or in a defined biological site within the animal.
An understanding of cytokine and CXC chemokine interactions and networks, as disclosed herein, allows for compositions other than the CXC chemokines themselves to be used in the present invention. This is the meaning of xe2x80x9ca composition that comprises or results in the production ofxe2x80x9d, as used herein. For example, IFNxcex3 results in the production of IP-10 and MIG, and could thus be used in the context of an angiostatic CXC chemokine. IL-10 decreases IP-10 and MIG levels (via decreasing IFNxcex3), and can thus be used similarly to angiogenic CXC chemokines.
xe2x80x9cCXC chemokine compositionsxe2x80x9d may comprise one or more CXC chemokine proteins, polypeptides or peptides. Equally, xe2x80x9cCXC chemokine compositionsxe2x80x9d may comprise one or more genes, nucleic acid segments or cDNAs that encode one or more wild type or engineered CXC chemokine proteins, polypeptides or peptides. As described hereinbelow, the coding segments or genes may be in the form of naked DNA, or housed within any one of a variety of gene therapy vehicles, such as recombinant viruses or cells, including tumor cells and tumor infiltrating lymphocytes, modified to contain and express the encoded CXC chemokine protein, polypeptide or peptide. The use of antisense CXC chemokine oligonucleotides is also contemplated.
As used herein, the term xe2x80x9cinhibiting angiogenesisxe2x80x9d is used in the same manner as the terms xe2x80x9cinducing or establishing angiostasisxe2x80x9d. This means that the process(es) of new or abnormal blood vessel growth (neovascularization) is reduced. xe2x80x9cNew blood vessel growthxe2x80x9d refers to the inappropriate growth of blood vessels, as may occur in disease states. The term xe2x80x9cnewxe2x80x9d describes blood vessels that are present in a number in excess of that observed in the normal state, this term does not represent the age of any particular vessel within a given individual.
Any consistently observed reduction of angiogenesis in response to the presence of a particular composition is evidence of the inhibition of angiogenic activity, and establishes the composition as a useful inhibitory or angiostatic composition. However, it will be understood that the most useful agents will result in a significant reduction in angiogenesis. xe2x80x9cA significant reduction in angiogenesis or angiogenic activityxe2x80x9d is defined herein as a consistently observed marked reduction or inhibition of angiogenesis, or the establishment of a significant angiostatic state.
Many systems are available for assessing angiogenesis. For example, as angiogenesis is required for solid tumor growth, the inhibition of tumor growth in an animal model may be used as an index of the inhibition of angiogenesis. Angiogenesis may also be assessed in terms of models of wound-healing, in cutaneous or organ wound repair; and in chronic inflammation, e.g., in diseases such as rheumatoid arthritis, atherosclerosis and idiopathic pulmonary fibrosis (IPF). It may also be assessed by counting vessels in tissue sections, e.g., following staining for marker molecules, e.g., CD3H, Factor VIII or PECAM-1.
Two systems are currently preferred by the present inventors for assessing angiogenesis. One is the endothelial cell chemotaxis assay. An angiogenic agent is identified in such an assay by acting to consistently promote endothelial cell chemotaxis above control values. In contrast to assays concerning neutrophil chemotaxis, inhibition of endothelial cell chemotaxis is evidence of anti-angiogenic activity. Anti-angiogenic agents can thus be identified by consistently reducing the endothelial cell chemotaxis back below the levels stimulated by an angiogenic agent. Such reductions are evidenced herein, e.g., in FIG. 11, FIG. 15, FIG. 22A-C, FIG. 30A, FIG. 30B, FIG. 31 and FIG. 34A-C.
The system most preferred by the present inventors for assessing angiogenesis is the corneal micropocket assay of neovascularization, as may be practiced using rat corneas. This in vivo model is widely accepted as being generally predictive of clinical usefulness. For example, as described in many review articles and papers such as those by O""Reilly et. al. (1994), Li et. al. (1991) and Miller et. al. (1994).
In the corneal micropocket assay, an angiogenic agent is an agent that consistently acts to promote the ingrowth of one or more blood vessels within the cornea, preferably without evidence of the influx of leukocytes. Most preferably, an angiogenic agent will be one that promotes the growth of a significant number blood vessels within the cornea. xe2x80x9cSignificantxe2x80x9d or xe2x80x9cpositive neovascularizationxe2x80x9d is herein defined as sustained directional ingrowth of capillary sprouts and/or hairpin loops towards a corneal implant that contains the test chemokine or substance. Many instances of angiogenesis in this model are shown herein, by way of example only, see FIG. 7A, FIG. 7B and FIG. 14B.
In the corneal micropocket neovascularization assay, a significant reduction in angiogenesis is evidenced by a consistently observed marked reduction in the number of blood vessels within the cornea. Such negative responses, which are evidence of angiostasis, are preferably defined as those corneas showing only an occasional sprout and/or hairpin loop that displayed no evidence of sustained growth when contacted with the test substance. For a xe2x80x9cmarked reductionxe2x80x9d to occur, it is not necessary that the number of blood vessels be reduced to zero, but rather that the blood vessel growth in the presence of a candidate inhibitory or angiostatic composition be much reduced in comparison to blood vessel growth in the absence of the inhibitor.
Examples of such inhibition of angiogenesis are evident by comparing FIG. 16F to FIG. 16B; FIG. 18B to FIG. 18E; FIG. 24A and FIG. 24B to FIG. 24E and FIG. 24F; and by comparing FIG. 25A and FIG. 25B to FIG. 25E and FIG. 25F. Inhibition of angiogenesis by MIG is particularly evidenced by comparing FIG. 29F to FIG. 29D, by comparing FIG. 29J to FIG. 29H and by comparing FIG. 29L to FIG. 29K, which show MIG inhibition of bFGF-, IL-8- and ENA-78-induced angiogenesis, respectively.
It is important to note that, in the present context, the inhibition of angiogenesis does not simply mean inhibition, antagonism or competition of one particular CXC chemokine. This is particularly exemplified with reference to IL-8. For example, the engineered angiostatic variant of IL-8 that contains TVR in place of ELR is capable of inhibiting angiogenesis induced, not only by native IL-8, but also by ENA-78 (FIG. 25A and FIG. 25B vs. FIG. 25E and FIG. 25F) and even by the non-CXC chemokine bFGF (FIG. 24A and FIG. 24B vs. FIG. 24E and FIG. 24F).
Further evidence of the newly discovered angiostatic properties of the CXC chemokines that do not contain the ELR motif is presented herein. For example, FIG. 14A-D show that IP-10 inhibits IL-8 induced angiogenesis; FIG. 16A-F show that IP-10 inhibits bFGF-induced angiogenesis; FIG. 18A, FIG. 18B and FIG. 18C show that IP-10 inhibits ENA-78-, GROxcex1, and GCP-2 induced angiogenesis, respectively. The ability of IP-10 to inhibit IL-8-induced angiogenesis is in contrast to its known failure to inhibit IL-8-induced neutrophil activation. Further studies of the inventors"" show that MIG inhibits not only ENA-78- and IL-8-, but also bFGF-induced angiogenesis.
Therefore, although a precise understanding of the mechanisms of action of the CXC chemokines is not necessary to practice the invention, it should be noted that both the natural and synthetic (engineered) non-ELR chemokines have definite angiostatic properties and do not function solely by competition or antagonism of a predominant angiogenic CXC chemokine, such as IL-8.
Certain currently preferred compounds for use in inhibiting angiogenesis are IP-10, MIG and CXC chemokines that have been modified to remove or mutate the ELR motif sequence. IP-10 may be purchased from Pepro Tech Inc. (Rocky Hill, N.J.), or may be prepared as described in any one of many published scientific articles, for example, Luster et al. (1985, 1987), and Luster and Ravetch (1987a, 1987b), each incorporated herein by reference.
MIG will most likely be prepared by expressing a nucleic acid molecule including a sequence as described by Farber (1993), incorporated herein by reference. A currently preferred method for preparing MIG is described in Example XIII and utilizes fusion protein production and subsequent cleavage.
The CXC chemokines that may be modified to remove the ELR sequence include IL-8, ENA-78, GCP-2, GROxcex1, GROxcex2, GROxcex3, CTAP-III, NAP-2 and xcex2TG. IL-8 may be purchased from many suppliers, e.g., R and D Systems, Minneapolis, Minn., or Genzyme; or may be prepared as described in any one of many published scientific articles, e.g., Schmid and Weissman (1987), Lindley et al. (1988), and Matsushima et al. (1988), each incorporated herein by reference. A currently preferred method is to prepare IL-8 as a recombinant fusion protein, and to cleave the protein to yield the IL-8 product (as described in Example XII).
ENA-78 may be purchased from R and D Systems. It may also be prepared using published methodology, such as described by, for example, Walz et al. (1991), Power et. al. (1994), Corbett et al. (1994) and Chang et. al. (1994), each incorporated herein by reference.
GROxcex1, GROxcex2 and GROxcex3 are available from R and D Systems, Minneapolis, Minn. and from Austral Biologicals, CA. The GRO genes and peptides may also be prepared as described in papers by, e.g., Anisowicz et al. (1988), Haskill et al. (1990), and Iida and Grotendorst (1990), each incorporated herein by reference.
CTAP-III, NAP-2 and xcex2TG may be prepared using the methodology of Walz et. al. (1989), which is incorporated herein by reference. The methods of Walz and Baggiolini (1990) and Begg et al. (1978), each incorporated herein by reference, may also be used to prepare CTAP-III, NAP-2 and xcex2TG. GCP-2 preparation may be achieved by the methods of Proost et. al. (1993a; 1993b), each incorporated herein by reference.
In addition to the foregoing, representative amino acid sequences of the various CXC chemokines are disclosed herein, as follows:
IP-10 amino acid sequence, SEQ ID NO:1;
MIG amino acid sequence, SEQ ID NO:2;
IL-8 amino acid sequence, SEQ ID NO:3;
ENA-78 amino acid sequence, SEQ ID NO:4;
GROxcex1 amino acid sequence, SEQ ID NO:5;
GROxcex2 amino acid sequence, SEQ ID NO:6;
GROxcex3 amino acid sequence, SEQ ID NO:7;
PBP amino acid sequence, SEQ ID NO:8;
CTAP-III amino acid sequence, SEQ ID NO:9;
xcex2TG amino acid sequence, SEQ ID NO:10;
NAP-2 amino acid sequence, SEQ ID NO:11; and
GCP-2 amino acid sequence, SEQ ID NO:12.
Several of the above sequences represent the CXC chemokine prior to processing. Using the following information, processed forms may be readily made. IP-10 is processed after Gly at position 21; MIG is processed after Gly at position 22. IL-8 is processed after Arg at position 27; ENA-78 is processed after Ser at position 36; GROxcex1 and GROxcex2 are processed after Gly at position 34; and GROY is processed after Gly at position 33. PEP is processed after Ala at position 34. Physiologically, further processing of PBP gives CTAP-III, xcex2TG and NAP-2, as represented by the above sequences. GCP-2 of SEQ ID NO: 12 may also be processed to give peptides with two, five and eight amino acids removed from the N-terminus.
It is important to note that each of the CXC chemokines are relatively short polypeptides. Each chemokine could thus be made using the presently available automated peptide synthesis technology. Smaller peptides could also be generated and then joined, resulting in the desired product.
The ELR triplet may simply be removed from the ELR-CXC chemokine compounds to create an angiostatic agent. However, in that various techniques of mutation or modification are known and easily practiced, it is currently preferable that the ELR motif be exchanged for a different amino acid triplet, most preferably one that occurs naturally in an angiostatic CXC chemokine. xe2x80x9cModified to removexe2x80x9d in the present application therefore encompasses both deletion and exchange or substitution. By changing the ELR amino acids rather than removing them, it is contemplated that the overall structure of the CXC chemokine will be less disturbed. Also, it is possible that the resultant modified CXC chemokine may be less immunogenic when administered to a patient.
A preferred example of a CXC chemokine that may be modified to remove the ELR sequence is IL-8 (also previously known as neutrophil-activating factor, monocyte-derived neutrophil-activating peptide, monocyte-derived neutrophil-chemotactic factor and neutrophil-activating peptide-1). Particular examples of modified IL-8 polypeptides are those in which the amino acid sequence ELR has been replaced with the amino acid sequence DLQ or the amino acid sequence TVR, both of which inhibit angiogenesis (see, e.g., FIG. 22A-C; FIG. 23D and FIG. 23E).
It is contemplated that virtually any combination of amino acids may be used to replace the ELR of an angiogenic CXC chemokine. However, any such mutant CXC chemokine generated should be tested to ensure that the anti-angiogenic activity is high enough to warrant progression to clinical practice. In preferred embodiments, the ELR motif will be exchanged for a triplet sequence that occurs in the naturally occurring angiostatic CXC chemokines. This is the origin of the amino acid sequences DLQ and TVR employed in the IL-8 mutants; with DLQ naturally being present in PF4 and TVR being found in IP-10. DLQ, TVR and KGR (from MIG) are thus preferred for replacing the ELR motif.
It will be understood that for human administration, the use of CXC chemokines purified from human cells or tissues, or recombinant CXC chemokines that comprise a sequence substantially as found in the human proteins and polypeptides, will generally be preferred. However, the use of CXC chemokines from other mammalian sources is by no means excluded.
In using the native or modified CXC chemokines in the invention, or even a gene that expresses such a chemokine, it is currently preferred to use the full-length protein or a gene that expresses such a molecule. However, it is also contemplated that shorter polypeptides or peptides will be effective in modifying angiogenesis or angiostasis, so long as the polypeptide or peptide contains the CXC motif and either the ELR motif or a substitution thereof. Therefore, biologically active fragments are encompassed by the invention.
Exemplary peptides that may be tested for use in inhibiting angiogenesis include VPLSRTVRCTC (SEQ ID NO:13) derived from IP-10; TPVVRKGRCSC (SEQ ID NO:14) derived from MIG; and even EAEEDGDLQCLC (SEQ ID NO:15) derived from PF4. Exemplary peptides that are envisioned for use in promoting angiogenesis include VPLSRELRCTC (SEQ ID NO:16), derived from IP-10; TPVVRELRCSC (SEQ ID NO:17) derived from MIG; and EAEEDGELRCLC (SEQ ID NO:18), derived from PF4.
CXC chemokine peptides of any length between about 8 or 9 amino acids and the length of the complete protein, or even longer, may be employed if desired. This includes peptides and polypeptides of about 10, 20, 30, 40, 50, about 100, or even about 150 amino acids in length. Additional peptidyl regions, may be added to the CXC chemokines if desired, and CXC chemokine-fusion proteins may also be used.
The CXC chemokine compositions for use in the invention may include proteins or peptides that have been modified or xe2x80x9cbiologically protectedxe2x80x9d. Biologically protected compositions, particularly peptides, have certain advantages over unprotected peptides when administered to human subjects and, as disclosed in U.S. Pat. No. 5,028,592 (incorporated herein by reference). Protected peptides therefore often exhibit increased pharmacological activity.
Compositions for use in the present invention may also comprise CXC chemokines that include all L-amino acids, all D-amino acids or a mixture thereof. The use of D-amino acids may be advantageous in certain embodiments, again particularly with peptides, as such peptides are known to be resistant to proteases naturally found within the human body, may be less immunogenic, and can therefore be expected to have longer biological half lives.
In certain embodiments, the angiostatic CXC chemokine will be administered to the animal or human subject by administering a composition that comprises a gene that expresses an XXX-ELR CXC chemokine other than PF4, as exemplified by IP-10, MIG and CXC chemokines that have been modified to remove the amino acid sequence ELR. As used herein, the term xe2x80x9cgene that expressesxe2x80x9d is used to refer to a gene, cDNA or other nucleic acid coding unit that encodes a particular CXC chemokine and that is capable of expressing the chemokine coding unit to produce the protein, polypeptide or peptide. cDNAs will generally be preferred over genomic sequences due to their ease of preparation and use.
The genes and cDNAs encoding the various CXC chemokines are well known to those of skill in the art. For example, IP-10 nucleic acid sequences are described in Luster et al. (1985) and Luster and Ravetch (1987b); and MIG nucleic acid sequences are described in Farber (1990), which concerns the mouse sequence, and Farber (1993), which concerns the preferred human sequence. Each of the foregoing being incorporated herein by reference.
IL-8 nucleic acid sequences are described in Lindley et al. (1988), Schmid and Weissmann (1987) and Matsushima et al. (1988); ENA-78 nucleic acid sequences are described in Walz et al. (1991), Power et. al. (1994), Corbett et al. (1994) and Chang et. al. (1994); and GROxcex1, GROxcex2 and GROxcex3 nucleic acid sequences are described in Anisowicz et al. (1988), Martins-Green et al. (1990, 1991), Iida and Grotendorst (1990), Richmond et al. (1988) and Haskill et al. (1990), each incorporated herein by reference.
CTAP-III, NAP-2 and RTG protein and nucleic acid sequences are highly related. An exemplary nucleic acid sequence is described in Wenger et. al. (1989); incorporated herein by reference. The GCP-2 amino acid sequence of Proost et. al. (1993b) and SEQ ID NO:12 may also be used to obtain GCP-2 cDNAs and genes, as described herein.
In addition, representative nucleic acid sequences of various CXC chemokines are disclosed herein. These include the IP-10 nucleic acid sequence of SEQ ID NO:89; the MIG nucleic acid sequence of SEQ ID NO:72; the IL-8 nucleic acid sequence of SEQ ID NO:77; and the ENA-78 nucleic acid sequence of SEQ ID NO:88. Further exemplary sequences included are the GROxcex1 nucleic acid sequence of SEQ ID NO:90; the GROxcex2 nucleic acid sequence of SEQ ID NO:91; the GROxcex3 nucleic acid sequence of SEQ ID NO:92; and the PBP nucleic acid sequence of SEQ ID NO:93 that results in CTAP-III, xcex2TG and NAP-2 sequences.
In using a nucleic acid segment that expresses a CXC chemokine, or that expresses an antisense CXC chemokine, the nucleic acid segment itself may be administered to the animal. This is based upon the knowledge that cells can take up naked DNA and express the encoded proteins or peptides, or the antisense mRNA. The utilization of this technology, and variations thereof, such as those described by Ulmer et al. (1993); Tang et al. (1992), Cox et al. (1993), Fynan et al. (1993), Wang et al. (1993), Gal et. al. (1993) and Whitton et al. (1993), each incorporated herein by reference, is therefore contemplated. The CXC chemokine DNA segments may be used in virtually any form, including naked DNA and plasmid DNA, and may be administered to the animal in a variety of ways, including parenteral, mucosal and gene-gun inoculations, as described, for example, by Fynan et al. (1993).
The use of recombinant viruses engineered to express CXC chemokines (or antisense versions thereof) is contemplated for use in the gene therapy embodiments. A variety of viral vectors, such as retroviral vectors, herpes simplex virus (U.S. Pat. No. 5,288,641, incorporated herein by reference), cytomegalovirus, and the like may be employed, as described by Miller (1992, incorporated herein by reference). Recombinant adeno-associated virus (AAV) and AAV vectors may also be employed, such as those described by Kotin (1994) and in U.S. Pat. No. 5,139,941, incorporated herein by reference. Recombinant adenoviral vectors are often used in the art and are particularly contemplated for use with the CXC chemokines. Techniques for preparing replication-defective infective viruses are well known, as exemplified by Ghosh-Choudhury and Graham (1987); McGrory et al. (1988); and Gluzman et al. (1982), each incorporated herein by reference. Liposome formulations are currently particularly preferred.
The compositions for use in the inhibitory methods described herein may contain only a single angiostatic CXC chemokine or CXC chemokine gene, or they may contain more than one such agent. The chemokine compositions may themselves be combined with other distinct angiostatic agents, or with other molecular entities such as antibodies, immunotoxins, Chemotherapeutic agents, and the like, as may be desired for use in the treatment of a particular disease or patient. Similarly, the treatment methods may be used alone or in conjunction with other modes of therapy.
In other embodiments, the invention provides further methods for the inhibition of angiogenesis, which methods generally comprise administering to an animal a biologically effective amount of a pharmaceutically acceptable composition that comprises one or more biological agents that inhibit an ELR-CXC chemokine other than IL-8, as exemplified by inhibiting one or more of ENA-78, GCP-2, CTAP-III, NAP-2 or xcex2TG. Such biological xe2x80x9cinhibitory agentsxe2x80x9d include antisense oligonucleotide constructs, polyclonal and monoclonal antibodies and, also, other molecular entities that function to inhibit ELR-CXC chemokines other than IL-8, and preferably, that inhibit one or more of ENA-78, GCP-2, CTAP-III, NAP-2 or xcex2TG genes, mRNAs or proteins.
CXC chemokine antisense oligonucleotide constructs will generally be designed to inhibit the transcription, translation or both, of a given CXC chemokine gene so that the level of the resultant protein product is reduced or diminished. Antisense oligos complementary to nucleic acid sequences of one or more of ENA-78, GCP-2, CTAP-III, NAP-2 and xcex2TG may be used to inhibit ELR-CXC chemokine gene expression in a given animal or human subject, thereby effecting the inhibition of angiogenesis. The antisense constructs may include antisense versions of CXC chemokine promoter, control region, exon, intron and/or exon:intron boundary sequences. The preparation of antisense oligos will be straightforward to those of skill in the art, given the details of the coding sequences in the present disclosure and in the published scientific literature.
In certain embodiments, one may wish to employ CXC chemokine antisense constructs that include other elements, for example, those which include C-5 propyne pyrimidines. oligonucleotides which contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression (Wagner et al., 1993).
Further biological inhibitory agents suitable for use in inhibiting ELR CXC chemokines, and angiogenesis, are polyclonal or monoclonal antibodies that bind to (have binding affinity for) and inhibit an ELR-CXC chemokine other than IL-8, as exemplified by ENA-78, GCP-2, CTAP-III, NAP-2 or xcex2TG. Polyclonal and monoclonal antibodies (MAbs) against CXC chemokines are available commercially, e.g., from R and D Systems. MAbs may also be generated using the well-established monoclonal antibody technology, which is known to those of skill in the art and is further described in the present disclosure. The more useful antibodies are contemplated to be those that neutralize at least between about 20 and about 30 ng of a particular CXC chemokine at a dilution of about 1:1000. In any embodiment involving antibodies, monoclonal antibodies, including humanized constructs, will be preferred.
The methods for inhibiting angiogenesis provided by the invention may be used in many contexts. For example, as angiogenesis is generally required for significant tumor growth, the anti-angiogenic strategies of the invention may be aimed specifically at attenuating tumor growth and/or metastasis. Typical vascularized tumors that may be treated using this invention include, but are not limited to, carcinomas and even sarcomas of the lung, breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate and thyroid; melanomas; gliomas; neuroblastomas; and the like. Non-small cell lung carcinomas (NSCLC), squamous cell carcinomas and adenocarcinomas are particularly suitable for treatment.
The anti-angiogenic methods may also be used to treat other diseases, including benign tumors, that rely on blood vessel growth. Such diseases particularly include hemangiomas, rheumatoid arthritis, atherosclerosis and idiopathic pulmonary fibrosis (IPF); but also include BPH, vascular restenosis, arteriovenous malformations (AVM), meningioma, neovascular glaucoma, psoriasis, angiofibroma, hemophilic joints, hypertrophic scars, osler-weber syndrome, pyogenic granuloma retrolental fibroplasia, scleroderma, trachoma, vascular adhesions, synovitis, dermatitis and even endometriosis. The levels of IL-8 and ENA-78 are herein shown to be elevated in hemangiomas.
Diseases and disorders associated with inappropriate blood vessel growth in the eye are particularly contemplated for treatment in accordance with the invention. These include pterygium, diabetic retinopathy and neovascularization associated with corneal injury or grafts.
IL-8 has been identified as at least one causal agent of injury in the adult respiratory distress syndrome (ARDS) (Miller et al., 1992); chronic bronchitis; rheumatoid arthritis (Brennan et al., 1990; Seitz et al., 1991; Koch et. al., 1991a); pseudogout (Miller and Brelsford, 1993); and cystic fibrosis (McElvaney et al., 1992; Nakamura et al., 1992; Bedard et al., 1993). It is therefore contemplated that the present invention may prove useful in treating one or all of the above diseases.
The potential use of IL-8 mutants and other non-ELR CXC chemokines to inhibit the inappropriate actions of IL-8 in other disease states, such as those above, is based upon the inventors"" assessment that these chronic diseases may have an angiogenic component. The invention is believed to modulate angiogenic-angiostatic responses, at least in part, through effects on endothelial cells. The invention is not particularly contemplated for use in modulating chemokine actions on neutrophils or in treating diseases that result solely from inappropriate neutrophil actions.
To treat any one of the above conditions, or any other disorder associated with increased angiogenesis, one would identify a patient having or suspected of having such a disease or disorder and administer to the patient an inhibitory composition comprising one or more XXX-ELR chemokine proteins or genes, other than PF4, including one or more ELR-CXC chemokine proteins or genes that has been modified to remove the amino acid sequence ELR. Equally, one may administer to the patient an inhibitory composition comprising one or more antibodies or antisense oligos directed against an ELR-CXC chemokine other than IL-8, such as being directed against ENA-78, GCP-2, CTAP-III, NAP-2 or xcex2TG. The inhibitory composition is administered in an amount effective to inhibit angiogenesis.
To reduce or control angiogenesis in this manner, one would generally administer a biologically effective amount of a pharmacologically acceptable inhibitory composition to an animal or human patient, in a manner effective to contact the area where inappropriate angiogenesis is occurring. Biological contact may be achieved simply by administering the composition to the animal or patient using virtually any pharmaceutical formulation and delivery method.
The formulation and delivery methods will generally be adapted according to the site of angiogenesis and the disease to be treated. Exemplary formulations include, but are not limited to, those suitable for parenteral administration, e.g., intravenous, intramuscular or subcutaneous administration, including formulations encapsulated in micelles, liposomes or drug-release capsules (active agents incorporated within a biocompatible coating designed for slow-release); ingestible formulations, e.g., for the treatment of gastric and duodenal ulcers; formulations for topical use, such as creams, ointments and gels; ophthalmic preparations; and other formulations such as inhalants, aerosols and sprays.
In certain embodiments, it will be preferred to inject the non-ELR angiostatic chemokine(s) directly into a localized site, such as a tumor site (intralesional injection) or joint. The procedures described in Sharpe et al. (1990) and Maione et al. (1991), each incorporated herein by reference, may be advantageously followed.
Various pharmaceutical compositions and techniques for their preparation and use will be known to those of skill in the art in light of the present disclosure. For a detailed listing of suitable pharmacological compositions and associated administrative techniques one may refer to the detailed teachings herein, which may be further supplemented by texts such as Remington""s Pharmaceutical Sciences, 18th ed., 1980, Mack Publishing Co., incorporated herein by reference.
The biologically effective amounts of the angiostatic CXC chemokines are considered to fall within a fairly broad range, in that low levels may be used to achieve at least some useful anti-angiogenic activity and higher levels used to achieve more significant biological effects. As disclosed herein, a variety of different chemokine concentrations proved effective in the corneal neovascularization assay, with a concentration of about 10 nM (between about 50 ng and about 80 ng) being particularly effective. Clinical doses that result in a local concentration of chemokine of about 10 nM are therefore contemplated to be particularly useful.
Naturally, in a clinical context, the amount of the CXC chemokine composition administered will depend on the host animal or patient, the condition to be treated and the route of administration. The precise amounts of active agent required to be administered will depend on the judgment of the practitioner and may be optimized to each individual by monitoring the biological effects and adjusting the dose accordingly.
Notwithstanding the fact that some dosage modification may be necessary, as is standard practice in the art, the determination of a suitable dosage range for use in humans will be straightforward in light of the data presented herein. For example, to inhibit angiogenesis doses of IP-10, MIG or an engineered XXX-CXC chemokine in the order of between about 0.1 mg/kg body weight (mg/kg) and about 10 mg/kg (including all integers between these values); per individual are contemplated.
In terms of antibodies that bind to, e.g., ENA-78, GCP-2, CTAP-III, NAP-2 or xcex2TG, effective amounts will be clinically equivalent amounts to the amount of IL-8 antibodies herein shown to reduce human tumor size and metastasis in a mouse model (FIG. 46; Table 6).
In further embodiments, the invention provides methods for stimulating angiogenesis. These methods generally comprise preparing CXC chemokine composition, preferably a pharmaceutically acceptable composition, that comprises, or results in the production of, an ELR-CXC chemokine other than IL-8, and administering the composition to an animal or human subject in an amount effective to stimulate angiogenesis. Natural chemokines, e.g., ENA-78, GCP-2, CTAP-III, NAP-2 and xcex2TG, and engineered CXC chemokines modified to contain the amino acid sequence ELR in the N-terminal region may be employed.
ENA-78, GCP-2, CTAP-III, NAP-2 AND xcex2TG, each of which contain the ELR motif, newly-identified as the angiogenic motif, will therefore be useful as angiogenic agents. These particular CXC chemokines have not been previously identified as having such angiogenic activities. CXC chemokines modified to contain the amino acid sequence ELR will generally be non-ELR chemokines into which the ELR sequence has been introduced. xe2x80x9cContainxe2x80x9d in this sense of containing the ELR sequence therefore encompasses CXC chemokines into which the ELR sequence has been added, and also those CXC chemokines that have been mutated or otherwise engineered to replace a distinct amino acid triplet with the ELR motif.
Particular examples of non-ELR CXC chemokines that may be modified to include the ELR motif are IP-10 and MIG. As shown in FIG. 45A and FIG. 45B, the inventors have modified MIG by the introduction of the ELR motif and shown that the resultant engineered chemokine has angiogenic activity (contrast with FIG. 45C and FIG. 45D).
Still further embodiments of the invention concern the stimulation of angiogenesis and wound healing by administering to an animal or patient a pharmaceutically acceptable composition that comprises an antibody that binds to and inhibits an XXX-CXC chemokine other than PF4; or an antisense oligonucleotide that binds to and inhibits an XXX-CXC chemokine other than PF4. It is currently preferred to inhibit IP-10 or MIG or an IP-10 or MIG gene or RNA. Such compositions will inhibit the angiostatic properties of these CXC chemokines, as newly-discovered, and will result in increased angiogenesis.
Following the inventors"" discovery of the function of the ELR motif in angiogenesis, it will be apparent that the teachings regarding the inhibition of angiogenesis described above can be readily applied to the stimulation of angiogenesis. Therefore human or other sources of CXC chemokines may be used; wild type, mutant, native, recombinant and synthetic ELR-CXC chemokines may be used, either alone or in combination; and the CXC chemokines may take the form of either proteins, polypeptides, peptides; or genes, nucleic acid segments, cDNAs, recombinant viruses or recombinant host cells that express ELR-CXC chemokine proteins, polypeptides or peptides.
Full-length or truncated ELR-CXC chemokines or their genes may be employed in these aspects of the invention, as may significantly shorter peptides. By way of example only, smaller peptides that are envisioned for use in stimulating angiogenesis include SAKELRCQC (SEQ ID NO:19) derived from IL-8; AGPAAAVLRELRCVC (SEQ ID NO:20) derived from ENA-78; DSDLYAELRCMC (SEQ ID NO:21) derived from CTAP-III; AELRCMCIKTTS (SEQ ID NO:22) derived from NAP-2; ESLDSDLYAELRCMC (SEQ ID NO:23) derived from xcex2TG; ASVATELRCQC (SEQ ID NO:24) derived from GROxcex1; APLATELRCQC (SEQ ID NO:25) derived from GROxcex2; ASVVTELRCQC (SEQ ID NO:26) derived from GROxcex3; and GPVSAVLTELRCTCLVRTLR (SEQ ID NO:27) derived from GCP-2. The ELR-CXC chemokine peptides may be biologically protected, and may include L-amino acids, D-amino acids or a mixture thereof.
The terms xe2x80x9cstimulating or eliciting angiogenesisxe2x80x9d mean that the process(es) of new blood vessel growth is enhanced. Any consistently observed increase in angiogenesis in response to the presence of a particular composition is evidence of angiogenic activity, and establishes the composition as a useful substance. CXC chemokines with significant angiogenic activity will often be preferred. xe2x80x9cSignificant angiogenic activityxe2x80x9d is defined herein as the establishment of angiogenesis in a previously silent system or model, or a consistently observed marked increase in angiogenesis. The corneal micropocket assay of neovascularization will again be a preferred model for assessing angiogenesis.
The stimulation of angiogenesis will generally be useful in the context of wound and sore healing. Other uses contemplated include, for example, in the treatment of vascular grafts and transplants and, particularly, in the treatment of skin, gastric and duodenal ulcers.
In still further embodiments, the invention provides methods for promoting wound-healing, which methods generally comprise contacting a wound or ulcer site of an animal with a biologically effective amount of a CXC chemokine composition comprising one or more ELR-CXC chemokines other than a GRO protein. IL-8, ENA-78, GCP-2, CTAP-III, NAP-2, xcex2TG and XXX-CXC chemokine polypeptides or peptides modified to contain the ELR motif may be used, as may genes that encode any of the foregoing. It is also contemplated that antibodies and antisense oligos that bind to the IP-10 and/or MIG (XXX-CXC chemokine) proteins and nucleic acids, respectively, may be employed in chronic wound healing.
The present invention particularly contemplates the use of the ELR CXC chemokines in the treatment of chronic wounds and ulcers. Although XXX-CXC chemokines are required in the overall wound-healing process, the inventors have discovered that the continued presence of non-ELR CXC chemokines correlates with chronic, non-healing wounds. Such wounds would thus benefit from the application of ELR CXC chemokines to redress the balance. ELR chemokines are herein shown to peak during natural wound healing (FIG. 9A-9F). It is contemplated that treatment with ELR CXC chemokines would be conducted for several days, with the dosage being reduced over time.
Although systemic administration is possible, local or directed administration of ELR-CXC chemokines is preferred for use in wound-healing. The chemokines may be added to the wound site, e.g., in the form of a cream, ointment, gel or lyophilized powder; formulated in an ingestible composition to reach an ulcer in the stomach or duodenum; or may be incorporated into a wound dressing that is applied to the wound site.
Biologically effective amounts will be those amounts that promote at least some wound-healing, with amounts that result in significant improvement of the healing process being preferred. The amount of the CXC chemokine compositions used in any given context will depend on the particular animal or patient and the site, type and degree of the wound or injury to be treated or healed. Notwithstanding the fact that some dosage modification may be necessary, as is standard practice in the art, doses of ELR chemokines of between about 0.1 mg/kg and about 10 mg/kg are again contemplated.
The invention also provides wound dressings, bandages and kits thereof that comprise one or more ELR-CXC chemokines, other than GRO proteins. IL-8, ENA-78, GCP-2, CTAP-III, NAP-2, xcex2TG and XXX-CXC chemokine polypeptides and peptides modified to contain the ELR motif are contemplated. ELR chemokines formulated into hydrocolloid dressings are particularly contemplated.
The invention also has important non-clinical uses, such as, for example, in bioassays of angiostasis and angiogenesis. By providing both positive and negative controls in assays of angiogenesis, the invention may be used to standardize such assays and provide control values against which the effectiveness of other candidate substances may be measured. By stimulating angiogenesis, the invention may also be used to promote tumor growth in experimental animals. Tumor-bearing animals are important tools in the development of anti-cancer drugs and strategies, therefore methods to increase the rate of tumor growth will be useful in providing target animals within a reduced time from inoculation with tumor cells.
In addition to providing many therapeutic and assay methods, the present invention also provides for certain diagnostic and prognostic methods. For example, the presence of an increased amount of one or more angiogenic CXC chemokines within a biological sample suspected of being prom a benign or malignant tumor, as compared to the amount of the CXC chemokine from a sample of the corresponding normal tissue, will often be indicative of the presence of such a tumor. The present examples demonstrate the novel finding that the levels of ENA-78 and GROxcex1 are elevated in benign and malignant tumors and that the level of the cytokine IL-10 (an indirect angiogenic agent) is increased in squamous cell carcinomas.
It is further contemplated that increased levels of the angiogenic ELR-containing CXC chemokines within or associated with a tumor will likely correlate with a more rapidly growing tumor and/or a tumor that is more likely to produce metastatic satellite tumors (see evidence in Table 6). Likewise, increased levels of the angiostatic non-ELR CXC chemokines will correlate with a less rapidly growing tumor and/or a tumor that is less likely to metastasize. Naturally, decreased ELR-CXC chemokine levels will be associated with less aggressive tumors and decreased XXX-CXC chemokine levels with more aggressive tumors. This also applies to the levels of other chemokines and cytokines, such as IL-10 and the interferons (IFNs) that act to modulate and regulate the levels of the CXC chemokines.
The invention thus further provides methods for characterizing a tumor, a preferred example of which comprises obtaining a sample from the tumor and testing the sample for the presence of one or more of GROxcex1, GROxcex2, GROxcex3, ENA-78, GCP-2, CTAP-III, NAP-2, xcex2TG or IL-10, wherein an increased amount one or more of the foregoing molecules is indicative of a tumor with increased angiogenic activity. IL-10 is indicative of angiogenesis by virtue of the fact that IL-10 decreases IFNxcex3, which in turn decreases IP-10 and MIG, and thus reduces the levels of the angiostatic CXC chemokines.
The use of diagnostic/prognostic tests using a panel, or plurality, of ELR CXC chemokines is contemplated to be particularly useful. Thus the sample will preferably be tested for the presence or increased levels of more than one of the ELR CXC chemokines or IL-10. For example, testing with one of GROxcex1, GROxcex2 or GROxcex3; in combination with one of CTAP-III, NAP-2 or xcex2TG; testing with IL-10 in combination with one of IL-8 or ENA-78; or testing with the entire panel of IL-8, GROxcex1, GROxcex2, GROxcex3, ENA-78, GCP-2, CTAP-III, NAP-2, xcex2RTG and IL-10 may be conducted. Defining the angiogenic activity within a given tumor in these ways is particularly useful as it will better allow appropriate treatment strategies to be defined and implemented.
The testing of the sample may take the form of testing for the presence of the particular protein, e.g., using antibody detection, or a biological assay of a fluid or tissue sample, e.g., using the endothelial cell chemotaxis or corneal micropocket assay of neovascularization. Testing may also take the form of testing for the presence of a nucleic acid sequence that encodes the CXC chemokine protein, e.g., using RT-PCR or nucleic acid hybridization technology.